Cancer immunotherapy with a viral antigen-defined, immunomodulator-secreting cell vaccine

ABSTRACT

A human cell line, which lacks major histocompatibility class I (MHC-I) antigens and major histocompatibility class II (MHC-II) antigens and which has been modified to comprise and express (i) a nucleotide sequence encoding an immunomodulator and (ii) a nucleotide sequence encoding a viral antigen, and a method of inducing or stimulating an immune response in a human to a viral-associated disease or cancer comprising administering to the human (i) the aforementioned human cell line in an amount sufficient to induce or stimulate an immune response to the viral associated disease or cancer, (ii) a human cell line, which lacks MHC-I and MHC-11 antigens and which has been modified to comprise and express a nucleotide sequence encoding an immunomodulator, and a human cell line, which lacks MHC-I and MHC-II antigens and which has been modified to comprise and express a nucleotide sequence encoding an antigen of EBV, simultaneously or sequentially in either order, by the same or different routes, in amounts sufficient to induce or stimulate an immune response to the viral-associated disease or cancer, or (iii) an immunomodulator and a human cell line, which lacks MHC-I and MHC-II antigens and which has been modified to comprise and express a nucleotide sequence encoding an antigen of EBV, simultaneously or sequentially in either order, by the same or different routes, in amounts sufficient to induce or stimulate an immune response to the viral associated disease or cancer.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of co-pending U.S. patent applicationSer. No. 10/528,311, filed Mar. 24, 2006, which is a U.S.Nationalization under 35 U.S.C. §371 of international application no.PCT/US03/29684, filed Sep. 19, 2003, which claims priority under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/411,990,filed Sep. 19, 2002, all of which applications are expresslyincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made, in part, as a result of funding from theNational Cancer Institute (NCI) Supplement through the University ofAlabama for work sponsored by the AIDS Malignancy Consortium, grant no.3U01CA70019-0751, from the NCI through Project 4, EBV Malignancies, BoneMarrow Transplantation in Human Disease, grant no. PO1 CA15396-28, andfrom the NCI through grant no. P50 CA 96888. Therefore, the U.S.government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to cancer immunotherapy and cancer vaccinedevelopment.

BACKGROUND OF THE INVENTION

Despite ongoing efforts to define immunologically relevant tumorantigens, very little is known about most tumor rejection antigens forthe majority of human cancers. Consequently, most cancer vaccineapproaches currently use tumor cells as a source of antigen. Earlygenerations of cell-based cancer vaccines have consisted of killed tumorcells or tumor cell lysates mixed with adjuvants, such as BacillusCalmette Guerin (BCG) and Corynebacterium parvum, in an attempt toamplify tumor-specific immune responses (Berd et al., J. Clin. Oncol. 8:1858-1867 (1990)). Subsequently, genetically modified tumor vaccinesreplaced the most complex and inconsistent mixtures of tumor cells andbacteria. Currently, the most popular genetically modified cell-basedvaccines take advantage of the large set of cloned genes encodingcytokines and co-stimulatory molecules (Pardoll, Ann. Rev. Immunol. 13:399-415 (1995)).

Among the different cytokines used to modify tumor immunogenicity,granulocyte-macrophage colony stimulating factor (GM-CSF) appears to bethe most potent (Dranoff et al., PNAS USA 90: 3539-3543 (1993)). GM-CSFinduces the differentiation of primitive hematopoietic precursors intodendritic cells (DC), a type of antigen-presenting cell (APC) thatinitiates the most potent T-cell responses (Banchereau et al., Nature392 (6673): 245-252 (1998)) and promotes DC recruitment anddifferentiation at the site of vaccination. Thus, DC play a central rolein priming immunological response.

GM-CSF-secreting cellular vaccines have been shown to eradicate small,pre-established tumors in mice (Dranoff et al., supra; and Levitsky etal., J. Immunol. 156: 3858-3865 (1996)). Furthermore, promising resultshave been obtained in human patients afflicted with melanoma (Soiffer etal., PNAS USA 95: 13141-13146 (1998)), prostate and renal cell carcinoma(Simons et al., Cancer Res. 59: 5160-5168 (1999); and Simons et al.,Cancer Res. 57: 1537-1546 (1997)), and pancreatic cancer (Jaffee et al.,J. Clin. Oncol. 19: 145-156 (2001)). These trials consistentlydemonstrated systemic anti-tumor immunity in patients and suggest animprovement in overall survival in those patients in whom evidence ofvaccine efficacy was demonstrated by the development of tumor-specificdelayed type hypersensitivity (DTH) responses (Jaffee et al., supra).

Unfortunately, modification of autologous tumor cells to express acytokine, such as GM-CSF, is highly individualized, expensive, andlabor-intensive. Therefore, simpler approaches that maintain theimmunological activity of paracrine cytokine production have beendeveloped. One such approach utilizes a universal bystander cell linealtered to produce a large and stable amount of GM-CSF (see, e.g.,Levitsky et al., U.S. Pat. No. 6,464,973 and Int'l Pat. App. No.PCT/US99/02253) in combination with an antigen of the cancer to betreated, such as, for example, tumor cells isolated from the patient(Borrello et al., Hum. Gene Ther. 10: 1983-1991 (1999); Borrello et al.,Blood 95: 3011-3019 (2000)). This approach obviates the need for invitro passaging or modification, such as by transduction, of eachpatient's tumor cells, thereby guaranteeing a constant amount ofcytokine production without any intra- or inter-patient variability,while utilizing the patient-specific antigenic repertoire. Anallogeneic, GM-CSF-secreting human erythroleukemia cell line, namelyK562, is currently being used in two phase I trials at Johns HopkinsUniversity for vaccination of multiple myeloma and acute myelogenousleukemia (AML), in combination with irradiated autologous tumor cells.

Vaccination of mice afflicted with lymphoma with a mixture of autologoustumor cells and GM-CSF-producing MHC class I- and MHC class II-negativecells, namely B78H1/GM-CSF cells, primed an anti-tumor immune response.The anti-tumor immune response was equivalent to or better than thoseachieved using autologous tumor cells directly transduced to secreteGM-CSF.

GM-CSF-secreting cellular vaccines, which are currently in use, are notspecific for a defined tumor antigen. Hence, it is not possible totarget such vaccines and evaluate fully their anti-tumor immuneresponses. It is an object of the present invention, therefore, toprovide a GM-CSF-secreting cellular vaccine that is specific for adefined tumor antigen. Such a vaccine will enable one to evaluate morefully anti-tumor immune responses. This and other objects and advantagesof the present invention, as well as additional inventive features, willbecome apparent from the detailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a human cell line, which lacks majorhistocompatibility class I (MHC-I) antigens and major histocompatibilityclass II (MHC-II) antigens and which has been modified to comprise andexpress (i) a nucleotide sequence encoding an immunomodulator and (ii) anucleotide sequence encoding an antigen of Epstein-Barr virus (EBV).Further provided is a composition comprising a human cell line, whichlacks MHC-I and MHC-II antigens and which has been modified to compriseand express a nucleotide sequence encoding an immunomodulator, and ahuman cell line, which lacks MHC-I and MHC-II antigens and which hasbeen modified to comprise and express a nucleotide sequence encoding anantigen of EBV. Still further provided is a composition comprising animmunomodulator and a human cell line, which lacks MHC-I and MHC-IIantigens and which has been modified to comprise and express anucleotide sequence encoding an antigen of EBV.

Also provided by the present invention is a method of inducing orstimulating an immune response in a human to an EBV-associated cancer.The method comprises administering to the human the aforementioned humancell line or one of the aforementioned compositions in an amountsufficient to induce or stimulate an immune response to the antigen ofEBV expressed by the human cell line, whereupon an immune response tothe EBV-associated cancer is induced. Alternatively, a human cell line,which lacks MHC-I and MHC-II antigens and which has been modified tocomprise and express a nucleotide sequence encoding an immunomodulator,and a human cell line, which lacks MHC-I and MHC-II antigens and whichhas been modified to comprise and express a nucleotide sequence encodingan antigen of EBV, can be administered, simultaneously or sequentiallyin either order, by the same or different routes, to the human inamounts sufficient to induce or stimulate an immune response to anEBV-associated cancer. Also, alternatively, an immunomodulator and ahuman cell line, which lacks MHC-I and MHC-II antigens and which hasbeen modified to comprise and express a nucleotide sequence encoding anantigen of EBV, can be administered, simultaneously or sequentially ineither order, by the same or different routes, to the human in amountssufficient to induce or stimulate an immune response to anEBV-associated cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a bi-cistronic plasmid (arrows indicate directionof transcription) containing the human GM-CSF gene (GM-CSF) operablylinked to the CMV promoter (CMV) and a polyA tail (polyA), a hygromycinresistance gene (Hygro.sup.r), an EBV EBNA1 gene (EBNA1), an EBV originof replication (OriP), and an ampicillin resistance gene (Amp.sup.r).The plasmid is designated pCEP4-EBNA1/hGM-CSF.

FIG. 2 is a diagram of a bi-cistronic plasmid (arrows indicate directionof transcription) containing the LMP2 coding sequence (LMP2) operablylinked to the CMV promoter (CMV) and a polyA tail (polyA), a neomycinresistance gene (Neomycin), and an ampicillin resistance gene(Amp.sup.r). The plasmid is designated pcDNA3-LMP2.

FIG. 3 is a compilation of various nucleotide (genomic, mRNA, cDNA;etc.) sequences that can be used in the context of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a human cell line, which lacks MHC-Iantigens and MHC-II antigens and which has been modified to comprise andexpress (i) a nucleotide sequence encoding an immunomodulator and (ii) anucleotide sequence encoding a viral antigen, in particular an antigenof a virus associated with a disease, such as cancer, e.g., EBV, humanpapilloma virus (HPV), or Kaposi sarcoma herpes virus (KSHV; also knownas human herpes virus 8 (HHV8)). The human cell line can be any suitablecell line. Theoretically, any human cell line that is capable ofparacrine production of an immunomodulator can be used. The capacity forparacrine production of an immunomodulator is not required when the cellline will not be used to express an immunomodulator in accordance withcertain embodiments of the present invention as set forth herein.

The cell line can naturally lack MHC-I and MHC-II antigens or it can bemanipulated or modified so that it does not express MHC-I and MHC-IIantigens. In this regard, it will be understood by one of ordinary skillin the art that a cell line is deemed to lack MHC-I and MHC-II antigensif it does not constitutively express such antigens under normalbiological conditions. However, a cell line that does not constitutivelyexpress MHC-I and MHC-II antigens can, under certain artificiallycreated conditions, such as conditions that can be created in vitro, beinduced to express MHC-I and/or MHC-II antigens. Such cell lines areconsidered to lack MHC-I and MHC-II antigens for purposes of the presentinvention. Likewise, cells having inactivated MHC antigens are alsoconsidered to lack such antigens for purposes of the present invention.

The cell line preferably grows in defined medium. One of ordinary skillin the art appreciates that defined medium is medium, the composition ofwhich is known. In other words, the identity and amount of each andevery component of the medium is known. Defined medium does not containserum inasmuch as the composition of serum is undefined. Preferably, thecell line grows as a suspension.

A preferred human cell line for modification in accordance with thepresent invention is K562, which is deposited with the American TypeCulture Collection (ATCC) as CCL-243. The K562 cell line is described byLozzio et al., Blood 45(3): 321-334 (1975), and Klein et al., Int. J.Cancer 18: 421-431 (1976). Other suitable human cell lines include, butare not limited to, SK-MEL-33 (Wang et al., J. Clin. Invest. 91: 684-692(1993)) and various melanoma cell lines (Ferrone et al., Immunol. Today16(10): 487-494 (1995); K. ageshita et al., Cancer Res. 53(14):3349-3354 (1993); and Wang et al., Tissue Antigens 47(5): 382-390(1996)).

A human cell line that expresses MHC-I antigens can be modified so thatit does not express such antigens in any of a number of different ways.For example, one can interfere with the expression and/or transport ofthe .alpha. chain. A human cell line that expresses MHC-II antigens alsocan be modified in various ways so that it does not express suchantigens. For example, one can interfere with the expression and/ortransport of the .alpha. chains and the .beta. chains. MHC-I and -IIantigens also can be inactivated for purposes of the present invention.This can be accomplished in a variety of ways (see, for example, U.S.Pat. No. 5,574,205). For example, a “dominant negative” can be created.A single modified .beta.sub.2 microglobulin gene, whose protein producteffectively complexes with MHC-I molecules and acts as a decoy, therebypreventing the insertion of MHC-I antigens into the membrane, can beoverexpressed. A similar approach can be used with respect to MHC-IIantigens by overexpressing modified genes encoding defective .alpha. or.beta. subunits that complex with the host cells' subunits, therebyrendering them nonfunctional. Transfection, retroviral infection orhomologous recombination can be used to achieve expression of modifiedMHC or .beta.sub.2 microglobulin genes or inactivation of genes.

Levels of MHC-I antigen on the cell surface can be reduced byintroducing into cells a sequence encoding adenoviral E19 protein bytransfection or retroviral infection. The protein forms complexesspecifically with MHC-I antigens in the rough endoplasmic reticulumpreventing normal transport of MHC-I molecules to the plasma membrane(Andersson et al., Cell 43: 215-222 (1985); and Pabo et al., Advances inCancer Research 42: 151-163 (1989)).

In addition to lacking MHC-I and MHC-II antigens, the human cell line ismodified to comprise and express a nucleotide sequence encoding animmunomodulator and a nucleotide sequence encoding a viral antigen. By“modified” is meant the introduction into the cell line of a nucleicacid molecule, e.g., a vector, comprising a nucleotide sequence encodinga gene product, which, in the context of the present inventive cellline, is an immunomodulator or an antigen, such as an antigen of EBV,HPV, or KSHV, in operable linkage with a promoter and, as required forexpression, various other regulatory sequences. Either theimmunomodulator is not expressed in the cell line or, as a result of theintroduction of the nucleic acid molecule is now expressed at a greaterlevel.

A “vector” encompasses a nucleic acid molecule, such as a plasmid, virusor other vehicle, which contains one or more heterologous or recombinantnucleotide sequences, e.g., a nucleotide sequence encoding animmunomodulator and/or a nucleotide sequence encoding an antigen of EBV,HPV or KSHV, wherein the nucleotide sequences can be under the controlof the same or different functional promoters, alone or in furthercombination with enhancer(s), and that is capable of functioning as avector as that term is understood by those of ordinary skill in the art.

Any suitable vector can be employed that is appropriate for introductionof nucleic acids into eukaryotic cells, or more particularly animalcells, such as mammalian, e.g., human, cells. Preferably, the vector iscompatible with the cell, e.g., can impart expression of theimmunomodulator and/or viral antigen, and is stably maintained orrelatively stably maintained in the cell. Desirably, the vectorcomprises an origin of replication. When an immunomodulator codingsequence or viral antigen coding sequence is transferred (i.e., asopposed to an immunomodulator gene having its own promoter or a viralantigen gene having its own promoter), optimally the vector alsocontains a promoter that can drive expression of the coding sequence andthat is operably linked to the coding sequence. A coding sequence is“operably linked” to a promoter (e.g., when both the coding sequence andthe promoter together constitute a native or recombinant immunomodulatorgene or viral antigen gene) when the promoter can direct transcriptionof the coding sequence.

Appropriate viral vectors include, but are not limited to simian virus40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpesvirus, vaccinia virus, Moloney murine leukemia virus, Harvey murinesarcoma virus, murine mammary tumor virus, and Rous sarcoma virus. Anyplasmid suitable for use in a eukaryote, in particular a mammal, e.g., ahuman, can be used in the context of the present invention. Desirably,the plasmid comprises a promoter, such as the cytomegalovirus promoter,an origin of replication, such as the SV40 origin of replication, aselectable marker, such as antibiotic resistance, and provides for mRNAwith poly A tails. A preferred example of a plasmid is pCEP4 (SeeExamples 1 and 3).

Reference to a vector or other DNA sequences as “recombinant” merelyacknowledges the linkage of DNA sequences, which are not typicallyconjoined as isolated from nature. A “gene” is any nucleic acid sequencecoding for a protein or a nascent mRNA molecule. Whereas a genecomprises coding sequences and non-coding (e.g., regulatory) sequences,a “coding sequence” does not include any non-coding DNA. As used herein,“gene” or “coding sequence” includes genomic or cDNA sequences, greaterand lesser sequences and mutations thereof, whether isolated from natureor synthesized in whole or in part, as long as the gene or codingsequence can express a protein having the characteristic function of theimmunomodulator, i.e., the ability to stimulate the host immuneresponse, or the characteristic antigenicity of an antigen of a virus.The means of modifying genes or coding sequences are well-known in theart, and also can be accomplished by means of commercially availablekits (e.g., New England Biolabs, Inc., Beverly, Mass.; Clontech, PaloAlto, Calif.).

A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis. “Enhancers” arecis-acting elements of DNA that stimulate or inhibit transcription ofadjacent genes. An enhancer that inhibits transcription also is termed a“silencer.” Enhancers differ from DNA-binding sites forsequence-specific DNA binding proteins found only in the promoter (whichalso are termed “promoter elements”) in that enhancers can function ineither orientation, and over distances of up to several kilobase pairs(kb), even from a position downstream of a transcribed region.

The “immunomodulator” can be any suitable immunomodulator, such as acytokine, a chemokine or an adjuvant, for example, obtained from anysuitable source, such as a mammal, e.g., a human. Desirably, theimmunomodulator induces or stimulates an immune response to the viralantigen expressed by the cell line. Cell-targeting means also can beconsidered immunomodulators Likewise, antibodies (or antigenicallyreactive fragments thereof), antisense molecules, dsRNAi, and the likealso can be considered immunomodulators to the extent that they inhibitor block the ability of a viral gene product to block the action of aninterferon, if so desired. For example, the EBNA-2 protein of EBV blockssignal transduction of interferons, the EBER RNA of EBV blocksactivation of Ph, and BCRF1 of EBV is an IL-10 homolog that inhibitsIFN-.gamma., IL-1, IL-2, and TNF synthesis.

Examples of suitable immunomodulatory cytokines include interferons(e.g., IFN.alpha., IFN.beta. and IFN.gamma.), interleukins (e.g., IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-20),tumor necrosis factors (e.g., TNF.alpha. and TNF.beta.), erythropoietin(EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1.alpha.,MIP-1.beta., Rantes, macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF), andgranulocyte-macrophage colony stimulating factor (GM-CSF), as well asfunctional fragments of any of the foregoing. The most preferredimmunomodulatory cytokine is GM-CSF, such as human GM-CSF, including afunctional fragment thereof. An alternatively preferred immunomodulatorycytokine is IL-2 or a functional fragment thereof. Any immunomodulatorychemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, orCX3C chemokine receptor, can be used in the context of the presentinvention. Examples of chemokines include, but are not limited to,Mip1.alpha., Mip-1.beta., Mip-3.alpha. (Lax), Mip-3.beta., Rantes,Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc,Elc, I309, IL-8, Gcp-2 Gro-.alpha., Gro-.beta., Gro-.gamma., Nap-2,Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well asfunctional fragments of any of the foregoing. Examples of adjuvantsinclude, but are not limited to, heat shock protein, CpG, Listeriamonocytogenes, aluminum hydroxide (for use with soluble antigen),aluminum phosphate (alum; for use with soluble antigen), muramyldipeptide, muramyl tripeptide, Mycobacterium tuberculosis, QuilA (apurified saponin from the plant Quillaja saponaria), alone or in furthercombination with glycosides, cholesterol, and/or phospholids, emptyadenoviral capsids; etc. One of ordinary skill in the art willappreciate that some of these adjuvants cannot be expressed from avector, in which case the adjuvant, when used, in combination with oneor more cell lines as described herein, is administered simultaneouslyor sequentially, in any order, with the one or more cell lines.Preferably, the adjuvant is administered with theviral-antigen-expressing cell line or the defined viral antigen, itself.Preferably, however, the immunomodulator nucleotide sequence encodes aGM-CSF sequence, particularly a human GM-CSF gene or coding sequence,including a human GM-CSF cDNA (e.g., as described by Cantrell et al.,PNAS USA 82: 6250-6254 (1985)) or genomic sequence (e.g., as describedby Miyatake et al., EMBO J. 4(10): 2561-2568 (1985)).

The viral antigen can be any defined antigen of a virus that isassociated with a disease, such as cancer, in a human. For example, theviral antigen can be any defined antigen of an oncogenic virus.Oncogenic viruses include, but are not limited to, RNA viruses, such asFlaviviridae and Retroviridae, and DNA viruses, such as Hepadnaviridae,Papovaviridae, specifically papillomaviruses, Adenoviridae,Herpesviridae, and Poxviridae. Desirably, the viral antigen is one towhich an immune response can be induced or stimulated in a human and isuniversally recognized. Preferably, the antigen is from EBV, HPV, orKSHV. Examples of suitable EBV antigens for expression in a human cellline in accordance with the present invention are described, forexample, in Herbst et al., PNAS USA 88: 4766-4770 (1991). Preferredantigens of EBV include, but are not limited to, Epstein-Barr nuclearantigen-1 (EBNA1), latent membrane protein 1 (LMP1), or latent membraneprotein 2 (LMP2). LMP2 is an especially preferred antigen of EBV. A cellline that expresses an antigen of EBV, in particular EBNA1, LMP1 orLMP2, can be used to induce or stimulate an immune response in a humanto an EBV-associated disease or cancer. In the event that immuneresponses are to be measured in accordance with methods set forthherein, preferably the EBV antigen is one that results in a CD8+ T-cellresponse that can be readily/easily measured. Examples of suitable HPVantigens for expression in a human cell line in accordance with thepresent invention are described, for example, in Van Ranst et al.;Virology 190(2): 587-596 (1992); and Rho et al.; Virology 203(1):158-161 (1994). Preferred antigens of HPV include, but are not limited,E5, E6, and E7. Examples of suitable KSHV antigens for expression in ahuman cell line in accordance with the present invention are described,for example, in Russo et al., PNAS USA 93(25): 14862-14867 (1996).Preferred antigens of KSHV include, but are not limited to, latencynuclear antigen (LANA) and v-cyclin.

Preferably, all proper transcription, translation and processing signals(e.g., splicing and polyadenylation signals) are correctly arranged onthe vector, such that the immunomodulator (viral antigen) nucleotidesequence will be appropriately transcribed and translated in the cellinto which it is introduced. The manipulation of such signals to ensureappropriate expression in host cells is well within the knowledge andexpertise of the ordinary skilled artisan. Whereas an immunomodulatorgene is controlled by (i.e., operably linked to) its own promoter,another promoter, including a constitutive promoter, such as, forinstance the adenoviral type 2 (Ad2) or type 5 (Ad5) major late promoter(MLP) and tripartite leader, the cytomegalovirus (CMV) immediate earlypromoter/enhancer, the Rous sarcoma virus long terminal repeat(RSV-LTR), and others, can be employed to command expression of theimmunomodulator coding sequence. The CMV promoter is a preferredpromoter. The same can also be said for the viral antigen gene.

Alternately, a tissue-specific promoter (i.e., a promoter that ispreferentially activated in a given tissue and results in expression ofa gene product in the tissue where activated) can be used in the vector.Such promoters include, but are not limited to, the elastase I genecontrol region, which is active in pancreatic acinar cells as describedby Swift et al., Cell 38: 639-646 (1984) and MacDonald, Hepatology 7:425-515 (1987); the insulin gene control region, which is active inpancreatic beta cells as described by Hanahan, Nature 315: 115-122(1985); the hepatocyte-specific promoter for albumin or.alpha.sub.1-antitrypsin described by Frain et al., Mol. Cell. Biol. 10:991-999 (1990), and Ciliberto et al., Cell 41: 531-540 (1985); and thealbumin and alpha.sub.1-antitrypsin gene control regions, which are bothactive in liver as described by Pinkert et al., Genes and Devel. 1:268-276 (1987), and Kelsey et al., Genes and Devel. 1: 161-171 (1987).

Similarly, a tumor-specific promoter, such as the carcinoembryonicantigen for colon carcinoma described by Schrewe et al., Mol. Cell Biol.10: 2738-2748 (1990), can be used in the vector. Along the same lines,promoters that are selectively activated at different developmentalstages (e.g., globin genes are differentially transcribed in embryos andadults) can be employed for gene therapy of certain types of cancer.

Another option is to use an inducible promoter, such as the IL-8promoter, which is responsive to TNF, or the 6-16 promoter, which isresponsive to interferons, or to use other similar promoters responsiveto other cytokines or other factors present in a host or that can beadministered exogenously. Use of a cytokine-inducible promoter has theadded advantage of allowing for auto-inducible expression of a cytokinegene. According to the invention, any promoter can be altered bymutagenesis, so long as it has the desired binding capability andpromoter strength.

Various methods can be employed for delivering a nucleic acid molecule,e.g., a vector, to a cell in vitro. For instance, such methods includeelectroporation, membrane fusion with liposomes, high velocitybombardment with DNA-coated microprojectiles, incubation with calciumphosphate-DNA precipitate, DEAE-dextran mediated transfection, infectionwith modified viral nucleic acids, direct microinjection into singlecells, and the like. Other methods are available and are known to thoseskilled in the art. The immunomodulator and the EBV antigen can beencoded on the same or different nucleic acid molecules.

If the cell line is to be used in the context of cancer immunotherapy,the immunomodulator desirably is one that induces or stimulates animmune response against a cancer cell or a cancer antigen, i.e., anyprotein, carbohydrate or other component that can elicit an immuneresponse, in particular, a defined viral antigen as expressed by a cellline in accordance with the present invention. An inhibitory cytokine ora cytokine that prevents priming cannot be used in the context of cancerimmunotherapy. While the nucleic acid molecule preferably encodes asingle immunomodulator, the nucleic acid molecule can encode two or moreimmunomodulators, which can be of the same type, e.g., both cytokines,such as cytokines that act synergistically, or of different types, e.g.,a cytokine and an adjuvant.

For purposes of identification and selection, preferably the nucleicacid molecule comprising a nucleotide sequence encoding animmunomodulator operably linked to a promoter and/or a nucleotidesequence encoding a viral antigen operably linked to a promoter furthercomprises a nucleotide sequence encoding a selectable marker operablylinked to a promoter. In other words, the nucleotide sequence encodingthe immunomodulator, the nucleotide sequence encoding the viral antigen,and the nucleotide sequence encoding the selectable marker can be on thesame nucleic acid molecule or on different nucleic acid molecules invarious combinations. Likewise, the nucleotide sequences can be underthe control of the same or different promoters.

Preferably, the selectable marker is an antibiotic resistance gene, suchas hygromycin resistance. When the selectable marker is hygromycinresistance, preferably the cell line is selected by growth in a culturemedium comprising at least about 400 .mu.g/ml hygromycin, morepreferably at least about 1,000 .mu.g/ml hygromycin.

A composition or implant, either one of which comprises anabove-described cell line and which is appropriate for administration invivo, can comprise appropriate carriers or diluents, which further canbe pharmaceutically acceptable. The means of making such a compositionor an implant have been described in the art, see, for instance,Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980). Use ofa balanced salt solution, such as Hanks' balanced salt solution, ispreferred in the composition.

Alternatively, the composition can comprise a human cell line, whichlacks MHC-I and MHC-II antigens and which has been modified to compriseand express a nucleotide sequence encoding an immunomodulator, and ahuman cell line, which lacks MHC-I and MHC-II antigens and which hasbeen modified to comprise and express a nucleotide sequence encoding anantigen of a virus that causes disease or cancer, such as an antigen ofEBV, HPV or KSHV. Also, alternatively, the composition can comprise animmunomodulator and a human cell line, which lacks MHC-I and MHC-IIantigens and which has been modified to comprise and express anucleotide sequence encoding an antigen of a virus that causes diseaseor cancer, such as an antigen of EBV, HPV or KSHV.

In pharmaceutical dosage form, a composition can be used alone or inappropriate association, as well as in combination, with otherpharmaceutically active compounds as are known in the art.

A composition of the present invention can be provided in unit dosageform, wherein each dosage unit contains a predetermined amount of thecomposition, alone or in appropriate combination with other activeagents. The term “unit dosage form” as used herein refers to physicallydiscrete units suitable as unitary dosages for human and other mammaliansubjects, each unit containing a predetermined quantity of thecomposition of the present invention, alone or in combination withanother active agent, calculated in an amount sufficient to produce thedesired effect, in association with a pharmaceutically acceptablediluent, carrier, or vehicle, where appropriate. The specifications forthe novel unit dosage forms of the present invention depend on theparticular pharmacodynamics associated with the pharmaceuticalcomposition in the particular host.

In view of the foregoing, the present invention also provides a methodof inducing or stimulating an immune response in a human to a virusassociated with a disease, such as cancer. The induction or stimulationof an immune response can be prophylactic or therapeutic and use of thephrase “inducing or stimulating” is intended to cover prophylactic andtherapeutic embodiments. For example, evidence is emerging that humanswho have had infectious mononucleosis are at risk for developingHodgkin's disease. Thus, the method of the present invention can be usedto inhibit the onset of a virus-associated disease or virus-associatedcancer/malignancy. In this regard, one of ordinary skill in the art willappreciate that, while prevention is desirable, “prophylactic” means anydegree in the inhibition of the onset of virus-associated disease orvirus-associated cancer inasmuch as any inhibition is beneficial.Likewise, one of ordinary skill in the art will appreciate that, whilecure is desirable, “therapeutic” means any degree ofinhibition/treatment of virus-associated disease or virus-associatedcancer, ranging from no change in the disease or cancer, which can bebeneficial inasmuch as the disease or cancer does not get worse, to alessening or an improvement of the disease or a reduction in cancer(size of a tumor and/or number of tumor) or an inhibition of metastasisof the cancer.

In particular, the present invention provides a method of inducing orstimulating an immune response to an EBV-associated disease or cancer,in particular an EBV-associated cancer. Likewise, the present inventionprovides a method of inducing or stimulating an immune response to aKSHV-associated disease or cancer, such as a KSHV-associated cancer, anda method of inducing or stimulating an immune response to anHPV-associated disease or cancer, in particular an HPV-associatedcancer. The method comprises administering to the human anabove-described human cell line in an amount sufficient to induce orstimulate an immune response to the virus-associated disease or cancer,e.g., malignancy. Upon administration of the cell line, an immuneresponse to the virus-associated disease or cancer, e.g., malignancy, isinduced or stimulated.

Alternatively, a human cell line, which lacks MHC-I and MHC-II antigensand which has been modified to comprise and express a nucleotidesequence encoding an immunomodulator, and a human cell line, which lacksMHC-I and MHC-II antigens and which has been modified to comprise andexpress a nucleotide sequence encoding an antigen of EBV, can beadministered, simultaneously or sequentially in either order, by thesame or different routes, to the human in amounts sufficient to induceor stimulate an immune response to an EBV-associated cancer. Also,alternatively, an immunomodulator and a human cell line, which lacksMHC-I and MHC-II antigens and which has been modified to comprise andexpress a nucleotide sequence encoding an antigen of EBV, can beadministered, simultaneously or sequentially in either order, by thesame or different routes, to the human in amounts sufficient to induceor stimulate an immune response to an EBV-associated cancer.

Examples of EBV-associated cancers/malignancies include Burkitt'slymphoma, T-cell lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma,B-cell lymphoma, gastric carcinoma, parotid carcinoma, breast carcinoma,and leiomyosarcoma. An example of an HPV-associated cancer/malignancy iscervical cancer. KSHV is associated with Kaposi's sarcoma, for example.

“Administering” means the actual physical introduction of thecomposition into or onto (as appropriate) the host. Any and all methodsof introducing the composition into the host are contemplated accordingto the invention; the method is not dependent on any particular means ofintroduction and is not to be so construed. Means of introduction arewell-known to those skilled in the art, and also are exemplified herein.

Any suitable route of administration can be used. Preferably, thecomposition is administered subcutaneously or intratumorally. Oneskilled in the art will recognize that, although more than one route canbe used for administration, a particular route can provide a moreimmediate and more effective reaction than another route. Local orsystemic delivery can be accomplished by administration comprisingapplication or instillation of the formulation into body cavities,inhalation or insufflation of an aerosol, or by parenteral introduction,comprising intramuscular, intravenous, intraportal, intrahepatic,peritoneal, subcutaneous, or intradermal administration. In the eventthat the tumor is in the central nervous system, the composition must beadministered intratumorally because there is no priming of the immunesystem in the central nervous system.

The amount of cells to be administered to induce or stimulate an immuneresponse to the defined viral antigen can be determined empirically(see, also, Examples 2 and 4 herein). For example, an initial low dosageof cells can be administered and the immune response to the definedviral antigen can be measured. If no immune response is induced orstimulated or it is deemed to be too low, the dosage of cells can beincreased. This process can be repeated every week or two weeks or sountil an effective dosage is administered.

One skilled in the art also is aware of means to monitor a therapeutic(i.e., systemic immune) response upon administering a composition of thepresent invention. In particular, the therapeutic response can beassessed by monitoring attenuation of tumor growth and/or tumorregression. The attenuation of tumor growth or tumor regression inresponse to treatment can be monitored using several end-points known tothose skilled in the art including, for instance, number of tumors,tumor mass or size, or reduction/prevention of metastasis. Methods ofassessing cervical cancer are described, for example, in U.S. Pat. No.6,388,064. These described methods are by no means all-inclusive, andfurther methods to suit the specific application will be apparent to theordinary skilled arts.

Any type of viral-associated cancer can be treated in accordance withthe present inventive method as long as an antigen of the virusassociated with the cancer has been defined and, desirably, is presenton the surfaces of the cancerous cells. “Cancer” as used herein includescancers, in particular those of epithelial origin, characterized byabnormal cellular proliferation and the absence of contact inhibition,which can be evidenced by tumor formation. The term encompasses cancerlocalized in tumors, as well as cancer not localized in tumors, such as,for instance, cancer cells that expand from a tumor locally by invasion.Thus, the method has applicability as a local adjuvant therapy forresected cancers as well as a local control of tumor growth.

The method of the present invention can be combined with other methodsof cancer treatment. Examples of such methods include radiation, surgeryand chemotherapy. In addition, the method of the present invention canbe adapted for non-human mammals, for example, by employing a nonhumanmammalian cell line and a non-human mammalian source of animmunomodulator and viral antigen, as appropriate.

The present inventive cell line has other uses, other than as describedabove. For example, the present inventive cell line can be used tocharacterize a human's immune response to the antigen, e.g., viralantigen, such as an EBV, HPV or KSHV antigen, expressed by the cellline. For example, if the antigen expressed by the cell line is an EBVantigen, the human's immune response to the EBV antigen can be measuredbefore and after administration of the cell line. By comparing theimmune responses before and after administration, it is possible todetermine whether or not a given human responds immunologically to theantigen of EBV and, if desired, characterize the nature and extent ofthe response. Tetramer assay and cytokine secretion assay can be used.If the human is to be treated for an EBV-expressing cancer, thisinformation can be used to determine if the human can be treated usingthe EBV antigen-expressing cell line. This information also can be usedto determine how best to administer the EBV antigen-expressing cellline, e.g., what dosage at what frequency. In much the same way, animmunocompromised human can be evaluated to determine suitability forcancer immunotherapy and, if found suitable, the manner of treatment,i.e., dosage and frequency of administration.

The present inventive cell line also can be used to assess an immuneresponse to a cancer cell vaccine for which the antigen is undefined.For example, the present inventive cell line can be administered to ahuman in combination with the cancer cell vaccine, and the presentinventive cell line can be used as a marker. The immune response to thedefined antigen expressed by the present inventive cell line can be usedto determine the human's immune responsiveness, thereby enabling gradingof immune responses to the cancer cell vaccine under similar vaccineconditions, for example.

In view of the teachings set forth herein, Applicants reserve the rightto pursue claims to the following embodiments. This reservation is notto be construed as a waiver of the right to pursue claims directed toother embodiments and modifications thereof as described herein.

A. A human cell line, which lacks MHC-I antigens and MHC-II antigens andwhich has been modified to comprise and express (i) a nucleotidesequence encoding an immunomodulator and (ii) a nucleotide sequenceencoding an antigen of HPV or KSHV.

B. The human cell line of A, wherein the antigen of HPV is E5, E6 or E7and the antigen of KSHV is LANA or v-cyclin.

C. The human cell line of A or B, wherein the immunomodulator is acytokine, a chemokine or an adjuvant.

D. The human cell line of C, wherein the cytokine is an inteferon, aninterleukin, a tumor necrosis factor, erythropoietin, FLT-3 ligand,macrophage colony stimulating factor (M-CSF), granulocyte colonystimulating factor (G-CSF), or granulocyte-macrophage colony stimulatingfactor (GM-CSF).

E. The human cell line of D, wherein the interferon (IFN) is IFN.alpha.,IFN.beta. or IFN.gamma.

F. The human cell line of D, wherein the interleukin (IL) is IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-8, IL-10 or IL-12.

G. The human cell line of D, wherein the tumor necrosis factor (TNF) isTNF.alpha. or TNF.beta.

H. The human cell line of C, wherein the chemokine is Mip1.alpha.,Mip-1.beta., Mip-3.alpha. (Lax), Mip-3.beta., Rantes, Hcc-1, Mpif-1,Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309,IL-8, Gcp-2 Gro-.alpha., Gro-.beta., Gro-.gamma., Nap-2, Ena-78, Gcp-2,Ip-10, Mig, I-Tac, Sdf-1, or Bca-1 (Blc).

I. The human cell line of C, wherein the adjuvant is a heat shockprotein or CpG.

J. The human cell line of A, wherein the immunomodulator is GM-CSF, andthe antigen of HPV is E6 or E7.

K. The human cell line of A or B, wherein the human cell line that ismodified is K562.

L. The human cell line of C, wherein the human cell line that ismodified is K562.

M. The human cell line of J, wherein the human cell line that ismodified is K562.

N. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of A or B in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

O. The method of N, wherein the human is female and has cervical cancer.

P. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of C in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

Q. The method of P, wherein the human is female and has cervical cancer.

R. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of J in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

S. The method of R, wherein the human is female and has cervical cancer.

T. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of K in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

U. The method of T, wherein the human is female and has cervical cancer.

V. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of L in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

W. The method of V, wherein the human is female and has cervical cancer.

X. A method of inducing or stimulating an immune response in a human foran HPV-associated cancer, which method comprises administering to thehuman the human cell line of M in an amount sufficient to induce orstimulate an immune response to the HPV-associated cancer, whereupon animmune response to the HPV-associated cancer is induced or stimulated.

Y. The method of X, wherein the human is female and has cervical cancer.

Z. A composition comprising a human cell line, which lacks MHC-I andMHC-II antigens and which has been modified to comprise and express anucleotide sequence encoding an immunomodulator, and a human cell line,which lacks MHC-I and MHC-II antigens and which has been modified tocomprise and express a nucleotide sequence encoding an antigen of HPV orKSHV.

AA. A composition comprising an immunomodulator and a human cell line,which lacks MHC-I and MHC-II antigens and which has been modified tocomprise and express a nucleotide sequence encoding an antigen of HPV orKSHV.

AB. A method of inducing or stimulating an immune response in a human toan HPV-associated or KSHV-associated cancer, which method comprisesadministering to the human the composition of Z (expressing an antigenof HPV or KSHV, respectively) in an amount sufficient to induce orstimulate an immune response to the HPV-associated or KSHV-associatedcancer, whereupon an immune response to the HPV-associated orKSHV-associated cancer is induced or stimulated.

AC. The method of AB, wherein the human is female and has cervicalcancer associated with HPV or the human has Kaposi's sarcoma.

AD. A method of inducing or stimulating an immune response in a human toan HPV-associated or KSHV-associated cancer, which method comprisesadministering to the human the composition of AA (expressing an antigenof HPV or KSHV, respectively) in an amount sufficient to induce orstimulate an immune response to the HPV-associated or KSHV-associatedcancer, whereupon an immune response to the HPV-associated orKSHV-associated cancer is induced or stimulated.

AE. The method of AD, wherein the human is female and has cervicalcancer associated with HPV or the human has Kaposi's sarcoma.

AF. A method of inducing or stimulating an immune response in a human toan HPV-associated or KSHV-associated cancer, which method comprisesadministering to the human a composition comprising a human cell line,which lacks MHC-I and MHC-II antigens and which has been modified tocomprise and express a nucleotide sequence encoding an immunomodulator,in an amount sufficient to induce or stimulate an immune response, andsimultaneously or sequentially, in either order, by the same route or adifferent route, a human cell line, which lacks MHC-I and MHC-IIantigens and which has been modified to comprise and express anucleotide sequence encoding an antigen of HPV or KSHV, respectively, inan amount sufficient to induce to stimulate an immune response,whereupon an immune response to the HPV-associated or KSHV-associatedcancer is induced or stimulated.

AG. The method of AF, wherein the human is female and has cervicalcancer associated with HPV or the human has Kaposi's sarcoma.

AH. A method of inducing or stimulating an immune response in a human toan HPV-associated or KSHV-associated cancer, which method comprisesadministering to the human an immunomodulator in an amount sufficient toinduce to stimulate an immune response, and simultaneously orsequentially, in either order, by the same route or a different route, ahuman cell line, which lacks MHC-I and MHC-II antigens and which hasbeen modified to comprise and express a nucleotide sequence encoding anantigen of HPV or KSHV, respectively, in an amount sufficient to induceto stimulate an immune response, whereupon an immune response to theHPV-associated or KSHV-associated cancer is induced or stimulated.

AI. The method of AH, wherein the human is female and has cervicalcancer associated with HPV or the human has Kaposi's sarcoma

EXAMPLES

Antibody generation and purification, diagnostic platforms, cloningprocedures; etc., to the extent that they are not described herein, canbe found in references such as the following:

Sambrook et al., Molecular Cloning, A Laboratory Manual, Vols. I-III,1989, Cold Spring Harbor Laboratory Press, USA;

Harlowe and Lane, Antibodies: A Laboratory Manual, 1988 and 1998, ColdSpring Harbor Laboratory Press, USA; and

Ausubel et al., Current Protocols, 2001, John Wiley and Sons, Inc.

The following examples serve to illustrate the present invention and arenot intended to limit its scope in any way.

Example 1

This example describes the generation of an EBV antigen-specific,GM-CSF-secreting cellular vaccine.

The allogeneic human erythroleukemia cell line K562 was transfected witha plasmid containing human GM-CSF operably linked to the cytomegaloviral(CMV) promoter, a hygromycin resistance gene, and the Epstein-Barr virus(EBV) nuclear antigen-1 (EBNA1) gene, which is required for the functionof the plasmid origin of replication. The plasmid, designatedpCEP4-EBNA1/hGM-CSF, is shown in FIG. 1. Hygromycin-resistant cloneswere screened for the secretion of GM-CSF. A K562-EBNA1/GM-CSF cloneproducing over 2,000 ng of GM-CSF/10.sup.6 cells/24 hrs was selected.The high degree of expression of this clone minimizes the number ofcells needed for vaccination, while leaving the margin for efficacy wellabove the threshold of 36 ng/10.sup.6 cells/24 hrs. TheK562-EBNA1/GM-CSF clone also was determined to express EBNA1 by Westernblot using a monoclonal antibody to EBNA1.

The K562-EBNA1/GM-CSF clone was transfected with a plasmid expressinglatent membrane protein 2 (LMP2) under the control of a CMV promoter.The plasmid, which is designated pcDNA3-LMP2, also contains a neomycinresistance gene and is shown in FIG. 2. G418-resistant clones wereanalyzed for the expression of LMP2 by immunofluorescent staining with amonoclonal antibody to LMP2. A clone expressing high levels of LMP2 wasselected and designated K562-EBNA1/LMP2/GM-CSF. The clone has beendetermined to express high levels of LMP2 (of expected size) and GM-CSF,as well as EBNA1 RNA and LMP2 RNA. A master cell bank can be generatedand a clinical grade vaccine can be generated from the master cell bank.The cells can be irradiated, frozen under controlled conditions, andstored in liquid nitrogen in vials containing 3.3.times.10.sup.7 cells.

Example 2

This example describes an EBV antigen-specific, GM-CSF-secretingcellular vaccine for treating EBV+ tumors.

Patients with EBV+ Hodgkin's lymphoma (HL) or nasophaaryngeal carcinoma(NPC) at high risk for relapse after primary therapy or those withrelapsed or metastatic disease are treated. The patients are given afirst vaccination six weeks after the completion of primary therapies,such as chemotherapy, radiation, or a combination thereof. Follow-upvaccinations are given monthly thereafter for a total of fourvaccinations, spanning weeks 6 to 18 during immune reconstitution. Onthe day of vaccination, the cellular vaccine (K562-EBNA1/LMP2/GM-CSF) isremoved from the liquid nitrogen storage and rapidly thawed in a37.degree. C. water bath. Viability of the cellular vaccine is assessedby trypan blue exclusion, and the number of viable cells is used forcalculation of dosages. The patients are intradermally injected with atotal dose of 3.3.times.10.sup.7 cells per vaccination divided into 9injections of 3.6.times.10.sup.8 cells in a volume of 0.5 ml. Threeinjections spaced 5 cm apart will be placed on each anterior thigh andthe non-dominant arm. Patients are monitored for possible toxicities atthe site of vaccination. Systemic toxicities are assessed from paracrinesecretion of GM-CSF.

If desired, the generation and enhancement of LMP2/EBNA1-specific CD4and CD8 T cell responses to LMP2 and EBNA1 are assessed using a modifiedIFN-.gamma. ELISPOT assay, which utilizes dendritic cells infected withrecombinant vaccinia virus expressing LMP2 or EBNA1 as a stimulator.This abolishes the need to HLA-type each patient. Briefly, peripheralblood mononuclear cells (PBMCs) from patients are fractioned into CD4and CD8 cells by a magnetic cell separation (MACS) system. Purified CD4or CD8 cells are stimulated with dendritic cells transduced withrecombinant vaccinia vector encoding LMP2 or EBNA1 in multiscreenhemagglutinin (HA) plates coated with a monoclonal antibody toIFN-.gamma. (capture antibody) for 16-18 hrs. The plates are then washedand stained for IFN-.gamma. with an immunoperoxidase technique. TheIFN-.gamma.-positive spots are counted using a stereomicroscope. Atetramer assay also is used for HLA-A2, A11 and A24 patients in order tocorrelate with results from the IFN-Y ELISPOT assay.

Patients are compared at baseline and at six months or earlier aftercompletion of vaccination. The baseline cellular response to LMP2 isexpected to be less than about 50/million PBMCs. A vaccination isconsidered to be successful if the cellular response exceeds about200/million PBMCs.

Example 3

This example describes the generation of an HPV antigen-specific,GM-CSF-secreting cellular vaccine.

The allogeneic human erythroleukemia cell line K562 was transfected witha plasmid containing human GM-CSF operably linked to the cytomegaloviral(CMV) promoter, a hygromycin resistance gene, and the Epstein-Barr virus(EBV) nuclear antigen-1 (EBNA1) gene, which is required for the functionof the plasmid origin of replication. The plasmid, designatedpCEP4-EBNA1/hGM-CSF, is shown in FIG. 1. Hygromycin-resistant cloneswere screened for the secretion of GM-CSF. A K562-EBNA1/GM-CSF cloneproducing over 2,000 ng of GM-CSF/10.sup.6 cells/24 hrs was selected.The high degree of expression of this clone minimizes the number ofcells needed for vaccination, while leaving the margin for efficacy wellabove the threshold of 36 ng/10.sup.6 cells/24 hrs. TheK562-EBNA1/GM-CSF clone also was determined to express EBNA1 by Westernblot using a monoclonal antibody to EBNA1.

The K562-EBNA1/GM-CSF clone is transfected with a plasmid expressing E6under the control of a CMV promoter. The plasmid, which is designatedpcDNA3-E6, also contains a neomycin resistance gene. G418-resistantclones were analyzed for the expression of E6 by immunofluorescentstaining with a monoclonal antibody to E6. A clone expressing highlevels of E6 was selected and designated K562-EBNA1/E6/GM-CSF. A mastercell bank can be generated and a clinical grade vaccine can be generatedfrom the master cell bank. The cells can be irradiated, frozen undercontrolled conditions, and stored in liquid nitrogen in vials containing3.3.times.10.sup.7 cells.

Example 4

This example describes an HPV antigen-specific, GM-CSF-secretingcellular vaccine for treating HPV+ tumors.

Patients with HPV+ cervical cancer at high risk for relapse afterprimary therapy or those with relapsed or metastatic disease aretreated. The patients are given a first vaccination six weeks after thecompletion of primary therapies, such as chemotherapy, radiation, or acombination thereof. Follow-up vaccinations are given monthly thereafterfor a total of four vaccinations, spanning weeks 6 to 18 during immunereconstitution. On the day of vaccination, the cellular vaccine(K562-EBNA1/E6/GM-CSF) is removed from the liquid nitrogen storage andrapidly thawed in a 37.degree. C. water bath. Viability of the cellularvaccine is assessed by trypan blue exclusion, and the number of viablecells is used for calculation of dosages. The patients are intradermallyinjected with a total dose of 3.3.times.10.sup.7 cells per vaccinationdivided into 9 injections of 3.6.times.10.sup.8 cells in a volume of 0.5ml. Three injections spaced 5 cm apart will be placed on each anteriorthigh and the non-dominant arm. Patients are monitored for possibletoxicities at the site of vaccination. Systemic toxicities are assessedfrom paracrine secretion of GM-CSF.

If desired, the generation and enhancement of E6-specific CD4 and CD8 Tcell responses to E6 is assessed using a modified IFN-Y ELISPOT assay,which utilizes dendritic cells infected with recombinant vaccinia virusexpressing E6 as a stimulator. This abolishes the need to HLA-type eachpatient. Briefly, PBMCs from patients are fractioned into CD4 and CD8cells by MACS separation system. Purified CD4 or CD8 cells arestimulated with dendritic cells transduced with recombinant vacciniavector encoding E6 or EBNA1 in multiscreen HA plates coated with amonoclonal antibody to IFN-.gamma. (capture antibody) for 16-18 hrs. Theplates are then washed and stained for IFN-.gamma. with animmunoperoxidase technique. The IFN-.gamma.-positive spots are countedusing a stereomicroscope. A tetramer assay also is used for HLA-A2, A11and A24 patients in order to correlate with results from the IFN-.gamma.ELISPOT assay.

Patients are compared at baseline and at six months or earlier aftercompletion of vaccination. The baseline cellular response to E6 isexpected to be less than about 50/million PBMCs. A vaccination isconsidered to be successful if the cellular response exceeds about200/million PBMCs.

Example 5

This example describes the generation of a KSHV antigen-specific,GM-CSF-secreting cellular vaccine.

The allogeneic human erythroleukemia cell line K562 was transfected witha plasmid containing human GM-CSF operably linked to the cytomegaloviral(CMV) promoter, a hygromycin resistance gene, and the Epstein-Barr virus(EBV) nuclear antigen-1 (EBNA1) gene, which is required for the functionof the plasmid origin of replication. The plasmid, designatedpCEP4-EBNA1/hGM-CSF, is shown in FIG. 1. Hygromycin-resistant cloneswere screened for the secretion of GM-CSF. A K562-EBNA1/GM-CSF cloneproducing over 2,000 ng of GM-CSF/10.sup.6 cells/24 hrs was selected.The high degree of expression of this clone minimizes the number ofcells needed for vaccination, while leaving the margin for efficacy wellabove the threshold of 36 ng/10.sup.6 cells/24 hrs. TheK562-EBNA1/GM-CSF clone also was determined to express EBNA1 by Westernblot using a monoclonal antibody to EBNA1.

The K562-EBNA1/GM-CSF clone is transfected with a plasmid expressingLANA under the control of a CMV promoter. The plasmid, which isdesignated pcDNA3-LANA, also contains a neomycin resistance gene.G418-resistant clones were analyzed for the expression of LANA byimmunofluorescent staining with a monoclonal antibody to LANA. A cloneexpressing high levels of LANA was selected and designatedK562-EBNA1/LANA/GM-CSF. A master cell bank can be generated and aclinical grade vaccine can be generated from the master cell bank. Thecells can be irradiated, frozen under controlled conditions, and storedin liquid nitrogen in vials containing 3.3.times.10.sup.7 cells.

Example 6

This example describes an KSHV antigen-specific, GM-CSF-secretingcellular vaccine for treating KSHV+ tumors.

Patients with Kaposi sarcoma at high risk for relapse after primarytherapy or those with relapsed or metastatic disease are treated. Thepatients are given a first vaccination six weeks after the completion ofprimary therapies, such as chemotherapy, radiation, or a combinationthereof. Follow-up vaccinations are given monthly thereafter for a totalof four vaccinations, spanning weeks 6 to 18 during immunereconstitution. On the day of vaccination, the cellular vaccine(K562-EBNA1/LANA/GM-CSF) is removed from the liquid nitrogen storage andrapidly thawed in a 37.degree. C. water bath. Viability of the cellularvaccine is assessed by trypan blue exclusion, and the number of viablecells is used for calculation of dosages. The patients are intradermallyinjected with a total dose of 3.3.times.10.sup.7 cells per vaccinationdivided into 9 injections of 3.6.times.10.sup.8 cells in a volume of 0.5ml. Three injections spaced 5 cm apart will be placed on each anteriorthigh and the non-dominant arm. Patients are monitored for possibletoxicities at the site of vaccination. Systemic toxicities are assessedfrom paracrine secretion of GM-CSF.

If desired, the generation and enhancement of LANA-specific CD4 and CD8T cell responses to LANA is assessed using a modified IFN-.gamma.ELISPOT assay, which utilizes dendritic cells infected with recombinantvaccinia virus expressing LANA as a stimulator. This abolishes the needto HLA-type each patient. Briefly, PBMCs from patients are fractionedinto CD4 and CD8 cells by MACS separation system. Purified CD4 or CD8cells are stimulated with dendritic cells transduced with recombinantvaccinia vector encoding LANA or EBNA1 in multiscreen HA plates coatedwith a monoclonal antibody to IFN-.gamma. (capture antibody) for 16-18hrs. The plates are then washed and stained for IFN-IFN-.gamma. with animmunoperoxidase technique. The IFN-.gamma.-positive spots are countedusing a stereomicroscope. A tetramer assay also is used for HLA-A2, A1 1and A24 patients in order to correlate with results from the IFN-.gamma.ELISPOT assay.

Patients are compared at baseline and at six months or earlier aftercompletion of vaccination. The baseline cellular response to LANA isexpected to be less than about 50/million PBMCs. A vaccination isconsidered to be successful if the cellular response exceeds about200/million PBMCs.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of inducing or stimulating an immune response in a human toan EBV-associated cancer, which method comprises administering to thehuman an immunomodulator in an amount sufficient to induce to stimulatean immune response, and simultaneously or sequentially, in either order,by the same route or a different route, a human cell line, which lacksMHC-I and MHC-II antigens and which has been modified to comprise andexpress a nucleotide sequence encoding an antigen of EBV, in an amountsufficient to induce to stimulate an immune response, whereupon animmune response to the EBV-associated cancer is induced or stimulated,wherein the human cell line is K562.
 2. The method of claim 1, whereinthe human has or is at risk for Hodgkin's lymphoma.
 3. The method ofclaim 1, wherein the human has or is at risk for nasopharyngealcarcinoma.
 4. The method of claim 1, wherein the human has or is at riskfor gastric carcinoma, Burkitt's lymphoma, T-cell lymphoma, B-celllymphoma, parotid carcinoma, breast carcinoma, and leiomyosarcoma. 5.The method of claim 1, wherein the immunomodulator is a cytokine, achemokine or an adjuvant.
 6. The method of claim 5, wherein the cytokineis an interferon, and interleukin, a tumor necrosis factor,erythropoietin, and FLT-3 ligand.
 7. The method of claim 6, whereininterferon (IFN) is IFN.alpha., IFN.beta, or IFN.gamma.
 8. The method ofclaim 6, wherein the interleukin (IL) is IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-8, IL-10, IL-12 or IL-20.
 9. The method of claim 6,wherein the tumor necrosis factor (TNF) is TNF.alpha, or TNF.beta. 10.The method of claim 5, wherein the chemokine is Mip1.alpha.,Mip-1.beta., Mip-3.alpha. (Larc), Mip-3.beta., Rantes, Hcc-1, Mpif-1,Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tare, Elc, I309,IL-8, Gcp-2 Gro-a, Gro-.alpha., Gro-.beta., Nap-2, Ena-78, Gcp-2, Ip-10,Mig, I-Tac, Sdf-1, or Bca-1 (Blc).
 11. The method of claim 5, whereinthe adjuvant is a heat shock protein or CpG.